How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols

Background Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic symptoms. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs for disease modelling remain key challenges to study human nociception in vitro. Here, we report a detailed characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol (“Anatomic” protocol) compared to the most commonly used small molecule approach (“Chambers” protocol). Methods Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Expression profiling of sensory neurons was performed with Immunocytochemistry and in situ hybridization techniques. Manual patch clamp and high throughput cellular screening systems (Fluorescence imaging plate reader, automated patch clamp and multi-well microelectrode arrays recordings) were applied to functionally characterize the generated sensory neurons. Results The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. High throughput systems confirmed functional expression of Na+ and K+ ion channels. Multi-well microelectrode recordings display spontaneously active neurons with sensitivity to increased temperature indicating expression of heat sensitive ion channels. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. Conclusions We validated the efficiency of two differentiation protocols and their potential application for understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

Background
Human induced pluripotent stem cell (iPSC)-derived sensory neurons offer a powerful tool to study cellular and molecular mechanisms of human disorders.Like embryonic stem cells, iPSCs are pluripotent, have the potential for self-renewal, and can differentiate into any adult cell type, while they have, in contrast to embryonic cells, the advantage that they can be generated from adult humans with their consent [1].During embryonic development of vertebrates, neural crest cells (NCCs) originating from the dorsal embryonic neural tube delaminate and migrate into the periphery.These multipotent NCCs further differentiate into several cell types including cells of the peripheral nervous system (PNS), melanocytes, cranial cartilage and bone, neuroendocrine cells, and several other phenotypes [2].
Peripheral sensory neurons with cell bodies located in the dorsal root ganglia (DRG) are a heterogeneous population of neurons that are involved in the detection and transmission of neural signals from the periphery to higher brain areas.Nociceptors respond to noxious thermal, mechanical, and chemical stimuli and can be further classi ed as A-bers (myelinated, large ber and large soma diameter) or Cbers (unmyelinated, small ber and small soma diameter).Dysfunction or neurodegeneration of sensory neurons can result in opposite clinical phenotypes ranging from pain insensitivity to severe episodic pain or painful chronic neuropathies [3].
Since the successful derivation of sensory neurons from iPSCs by Chambers and colleagues [4], considerable progress has been made in generating neural lineages that can be differentiated into peripheral sensory neurons [5][6][7][8][9].However, current iPSC differentiation protocols vary widely in e cacy and most likely do not generate the full diversity of nociceptor populations.Differences in the e cacy and reproducibility of differentiation also depend on genetic variations in cell lines, initial density, culture conditions at the start of the experiment, minor differences in small molecules, and coating conditions [10].It has been reported that small changes in culture conditions and differentiation protocols can signi cantly change the resulting transcriptome making comparisons between labs and thus reproducibility di cult [11].It has been shown that iPSC-derived from different somatic cell types result in varied differentiation potential e ciency [12].These factors remain a key challenge for the generation of subtype-speci c neurons from iPSCs.When all these variables are considered, it is often necessary to customize iPSC line speci c sensory neuron differentiation protocols to improve yields and e ciencies for different lines grown under different conditions.However, consistent differentiation protocols, which are both highly e cient and rapid, would be bene cial for the generation of functional subtype speci c sensory neurons, or a more physiological mixture of subtypes, for human disease modelling.iPSC-derived from patients suffering from genetic pain syndromes offer the unique possibility to study the effect of genetic variations directly on the affected cells, the patient's nociceptors [10].For example, gain-of-function mutations in the SCN9A gene encoding voltage gated sodium ion channel Nav1.7 have been shown to cause Inherited Erythromelalgia (IEM).IEM is a chronic pain condition that results in intense burning pain of extremities often triggered by warming of the affected limbs.Chronic neuropathic pain associated with Small Fiber Neuropathy (SFN) often manifests with intense burning pain in the peripheral limbs and is accompanied by elevated temperature detection thresholds and often by decreased epidermal nerve ber density [13,14].Several mutations have been linked to Nav1.7 and Nav1.8 ion channels causing SFN [15,16].This renders voltage-gated sodium ion channels, particularly Nav1.7 and Nav1.8, as attractive therapeutic targets for pain disorders.Unfortunately, most of the selective Nav1.7 and Nav1.8 inhibitors have shown limited clinical e cacy so far [17][18][19].iPSC-derived sensory neurons as a disease model system have been used to study IEM [20][21][22] and SFN disorders [23] providing valuable insight into rare genetic forms of pain.Cao et al., 2016 22 showed the cellular disease phenotype and its reversal through selective Nav1.7 blockade using iPSC-derived sensory neurons from patients with IEM disorder.iPSC-derived sensory neurons helped to identify an FDA-approved drug in vitro as an effective treatment option for a patient suffering from SFN, highlighting the potential of iPSCderived sensory neurons as a model system to study neurological disorders [23].
Here, we compare two iPSC differentiation protocols: the widely used "Chambers" protocol [4] and the protocol from Anatomic Incorporated (herein referred to as the "Anatomic protocol") which uses a standard differentiation kit, Senso-DM, inducing a naive early ectodermal intermediate cell type using combined inhibition of bone morphogenic protein and broblast growth factor signalling pathways [24].This Anatomic protocol leads to an accelerated differentiation of iPSCs into immature neurons within Day 7 of differentiation, whereas functional mature neurons emerge from both protocols at Day 28.Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons including Anatomic's commercially available RealDRG™ iPSC-derived sensory neurons.We functionally characterized the generated neurons at different days of maturation.iPSC-derived from two patients suffering from IEM and SFN chronic pain conditions were used for disease modelling with the Anatomic protocol.Diseased iPSC-derived neurons displayed cellular hyperexcitability and reduced rheobase as compared to control groups, indicating that this protocol may also be used for modelling disease in a dish.We compare the utility of this protocol to the more standard Chambers protocol for the generation of electrophysiologically mature and active neurons.

Methods iPSC lines from healthy and diseased subjects
In the present study iPSCs were derived from mesenchymal stromal cells (female 69 years) and broblasts (repository name CS00iCTR21n1; Male 6 years) of two healthy subjects designated as Ctrl1 and Ctrl2 respectively (Table 1).Ctrl2 has been referred to as a "di cult clone" in this study due to its lower differentiation potential with Chambers protocol in generating peripheral neuronal lineage (Fig. S1).
iPSC-derived from blood cells of a female 9-year-old suffering from IEM a phenotypically similar but different patient to the rst reported by Skeik et al., 2012 25 and broblasts from a female 69-year-old suffering from SFN disorder (repository name UKERi313-R1, [23] were used for disease modelling designated in this study as IEM and SFN, respectively (Table 1).iPSCs generated from blood cells of the IEM patient were found to be heterozygous for the p.Q875E mutation in the Nav1.7 ion channel (Fig. S2).
Table 1: Clinical phenotype of subjects used in the study iPSC-derived sensory neurons from Anatomic (RealDRG™) For membrane potential FLIPR assay, multielectrode arrays, automated patch clamp and in-situ hybridization studies, Anatomic's commercially available iPSC-derived sensory neurons (RealDRG™) were used (Table 1).These neurons were manufactured with scaled-up versions of Anatomic's Senso-DM kit.
The iPSC line derived from female subject ANAT001 was maintained under fully-de ned conditions before seeding onto a de ned matrix.Cultures were fed optimized differentiation formulations daily to produce immature sensory neurons by day 7 post-induction.Day 7 cultures were dissociated and cryopreserved.Lot-speci c metrics were recorded including yield, cell number per vial, viability, post-thaw recovery, post-thaw viability, post-thaw morphology, purity, and sterility.Criteria used to determine lots passing quality control included neuronal purity > 95%, veri ed cell number per vial, post-thaw viability > 70%, and sterility.Lots passing quality control were shipped on dry ice to end users and used according to the manufacturer's instructions.Additional detail related to differentiation, media compositions, materials used, and bioprocessing steps are proprietary information of Anatomic.

Differentiation of iPSCs to sensory neurons
Differentiation of all iPSCs (healthy and diseased) was carried out using Senso-DM (Anatomic Incorporated, cat# 7007) and Chambers et al., 2012 4 with modi cations designated as "Chambers protocol" in this study.All clones were differentiated once, except Ctrl1 iPSCs which were differentiated twice for this study.

Anatomic differentiation protocol
The Anatomic differentiation protocol was used for all cell lines investigated in the study.Two variations of the differentiation were compared, one utilizing single cell seeding and one utilizing clump seeding.Ctrl1 iPSCs were differentiated with two different seeding protocols.All other iPSC lines were differentiated with single cell seeding protocol.An optimal seeding density was determined for each cell line with both single cell and clump seeding protocols to maximize yield and e ciency.For clump seeding an ideal iPSC colony size was used ranging between 25 and 50 cells per colony.iPSC cultures with a 60-80% con uence were used for plating the cells as either single cell or clump on Matrix1 (Anatomic Incorporated, cat# M8001) pre-coated wells.iPSCs were seeded as single cells in a density of 15,000-80,000 cells/cm 2 with 10 µM Y-27632 (Abcam Biochemicals, Bristol, United Kingdom).For both single cell and clump seeding protocols, a cocktail of small molecules was added from DIV0 (Days in vitro) through DIV7 of differentiation.Chrono™ Senso-DM 1, 2, 3, 4, 5, 6 and 7 (Small molecules, Anatomic Incorporated, cat# 7007) were added on each day of differentiation starting from DIV0.For each day of differentiation, 0.5 mL of Senso DM (1-7) supplement was added to 4.5 mL of Basecamp (Differentiation basal medium, Anatomic) to create 5 mL of complete differentiation medium that was fed immediately to cultures.Immature neurons generated on DIV7 were then dissociated using Accutase (Sigma cat# A6964), incubated at room temperature for ~ 1 hour which ensured isolation of single neurons.30,000-40,000 cells were then plated onto glass coverslips, coated with PDL 0.1mg/ml (Sigma cat# P0899) and Matrix 3 (Anatomic Incorporated, cat# M8003).Neurons were then supplemented with Chrono™ Senso-MM maturation medium (Anatomic cat# 7008) from DIV7 onwards.Two-thirds medium exchanges were performed thrice weekly.

Chambers differentiation Protocol
Ctrl1 cell line was differentiated with the Chambers protocol to compare the potential of differentiation e ciency and generation of sensory neurons to the Anatomic protocol.Ctrl1 iPSCs were differentiated following a previously published protocol with modi cations [4,10].Brie y, iPSCs were seeded as single cells in a density of 30,000-40,000 cells/cm 2 with 10 µM Y-27632 (Abcam Biochemicals, Bristol, United Kingdom).When cells reached 80-90% con uency, usually 24-48 h after plating, neural conversion was induced using dual-SMAD inhibition.LDN-193189 (1 µM, Sigma-Aldrich) and SB431542 (10 µM, Miltenyi Biotec) were added to the culture medium between DIV0-5.To accelerate neural crest speci cation and peripheral neuron formation from neural crest cells, three small molecules (3 µM CHIR99021, 10 µM DAPT, and 10 µM SU5402, all Tocris, United Kingdom) were added between DIV2-10.Between DIV0-5, cells were fed with knockout DMEM/F-12 containing 15% KnockOut serum replacement, 1 mM Lglutamine, 100 µM NEAA, 100 µM β-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin (all from Thermo Fisher Scienti c).Between DIV4-10, cells were fed with DMEM/F-12, containing 10 ml/l N2 (1X), 20ml/l B27 (1X) without vitamin A supplements and 100 U/ml penicillin, 100 µg/ml streptomycin (all from Thermo Fisher Scienti c).N2/B27 medium was added to basal medium at 25% between days 4-5, 50% between days 6-7 and 75% between days 8-10.The culture medium was changed daily.mOsm).Resting membrane potential (RMP) was recorded immediately after establishing the whole-cell con guration for 4 seconds.The rst action potential (AP) evoked by the square pulse protocol (increments of 10 pA) was used to identify the AP properties and maturity of neurons.All recorded neurons having APs with an overshoot above 0 mV were considered mature.The AP threshold was de ned as the potential at which the minimum of the rst derivative of the AP (the point of in ection during the depolarization) occurs.Afterhyperpolarization (AHP) was calculated as the minimum potential recorded during the repolarization phase of AP.The number of APs generated and time to 1st AP was determined in response to ramp current stimulus of 500 pA/500 ms.Depolarisation current ramps of 1 nA were given over 100-1000 ms to assess ring in response to slow depolarisation.For calculating the number of APs in response to ramp current injections, APs with an overshoot above 0 mV were counted as APs.After measurement in the current-clamp con guration, the ampli er was switched to the whole cell voltage-clamp mode to measure both sodium and potassium currents.Voltage dependence of activation for both Na + and K + currents were recorded with a 500 ms pulse from − 80 mV to 40 mV in 10 mV steps from a holding voltage of -90 mV.determined with a 100 ms pulse from − 90 mV to 40 mV in 10 mV increment steps from a holding voltage of -120 mV.

Voltage-clamp recordings
To assess the presence of Nav1.8 currents, 1µM of the selective Nav1.8 blocker A-887826 (provided by Grünenthal) was used on DIV31 of maturation from Ctrl1 iPSC-derived sensory neurons [29].A-887826 was dissolved in DMSO as 10 mM stock solution and the nal maximum concentration of DMSO was 0.1%.Solutions were applied through a gravity-driven perfusion system.A single pulse voltage protocol from a holding potential of − 120 mV to − 30 mV repeated every 10 s was used.Cells were patched in the presence of 500 nM TTX.After obtaining a stable baseline recording with 500 nM TTX, 1 µM A-887826 was applied to check the percent inhibition of currents.In one cell, washout with extracellular solution (ECS) was performed to obtain the total sodium currents at the end of the experiment.

In Situ Hybridization
RNAscope in situ hybridization was used to characterize expression of key nociceptor markers in RealDRG™.RealDRG™ iPSC-derived neurons were plated in 8-well chamber slides (Thermo Scienti c cat# 154534) coated with 0.1% PLO (Sigma-Aldrich cat# P4957)/Matrix 3 (Anatomic Incorporated) and maintained with Anatomic protocol instructions.In situ hybridization was completed using the RNAscope procedure (multiplex version 1 assay (320851)) utilizing manufacturer's (Advanced Cell Diagnostics ACD) published protocols [30].On DIV14 and DIV16, chambers were disassembled from the slide, and the cells were xed in 10% neutral buffered formalin for 30 minutes at room temperature.Slides were then washed twice in 1X PBS, and boundaries were drawn around each well using the hydrophobic ImmEdge PAP pen (Vector Labs cat# H-4000).Slides were washed again in 1X PBS before being incubated in protease III reagent (1:30 in 1X PBS) for 10 minutes at room temperature in a humidity control tray.Slides were washed twice in 1X PBS and then placed in a prewarmed humidity control tray with dampened lter paper to be incubated with probe mixtures for 2 hours at 40°C.DIV14 slides were incubated with Channel 1 NTRK1 (ACD cat# 402631), Channel 2 TAC1 (ACD cat# 310711), and Channel 3 HCN2 (ACD cat# 517021).DIV16 slides were incubated with Channel 1 SCN10A (ACD cat# 406291), Channel 2 TAC1 (ACD cat# 310711), and Channel 3 TRPV1 (ACD cat# 451381).Slides also had one well each for positive (ACD cat# 320861) and negative (ACD cat# 320871) control probes.Following probe incubation, slides were washed twice in 1X RNAscope wash buffer and incubated in AMP-1 reagent for 30 minutes at 40°C.
Washes and incubation were repeated for AMP-2, AMP-3, and AMP-4A for 15 minutes, 30 minutes, and 15 minutes, respectively.After ampli cation, slides were washed in 0.1M phosphate buffer (PB, pH 7.4) and stained with DAPI (Cayman Chemical cat# 14285).Slides were then washed twice in 0.1M PB, air dried, and cover-slipped with Prolong Gold Antifade (Fisher Scienti c cat# P36930) mounting medium.Images were acquired on an Olympus FV1200 confocal microscope using a 40X objective and analyzed using Cellsens software (Olympus).

FLIPR (high-throughput plate reader assays)
For high-throughput uorescent imaging assays using the FLIPR Penta (Molecular Devices), RealDRG™ were thawed and cultured in Senso-MM (Anatomic Incorporated).Cells were seeded at a density of 1,000-10,000 cells/well on black-walled 384-well imaging plates (Corning CLS3657) pre-coated with

Multi-well Microelectrode Arrays
Multi-well microelectrode arrays (MEA) were used to evaluate RealDRG™.The day before starting the culture, a 48-well MEA plate (Axion Biosystems, cat# M768-tMEA-48W) was coated with Poly-L-Ornithine (0.01%, EMD Millipore Sigma cat#A-004-C) and incubated overnight at room temperature.After 3 washes with sterile deionized water, each well was coated with a Matrix 3 (1:50 dilution with dPBS (-/-) from Anatomic, cat#M8003) and incubated for 3 hours at 37 °C.RealDRG™ were thawed and cultured in Senso-MM (Anatomic, cat#7008).Cells were resuspended in 2 mL of Chrono™ Senso-MM complete growth medium and counted for a seeding density of 40k cells/cm 2 .Immediately prior to seeding, excess Chrono™ Matrix 3 (from plate preparation step) was removed from each well.Cells were added to wells at a total volume of 400µl in Chrono™ Senso-MM.Chrono™ Senso-MM media was exchanged at 50% from a 400 µl volume every other day and cells were grown at 5% CO 2 at 37 °C.
MEA electrophysiology data were acquired from an Axion Maestro Classic system at 12.5 kHz sampling rate and processed with a single pole Butterworth bandpass lter (300-5000 Hz).Individual spikes were detected from ltered continuous voltage recordings where exceeding ± 5.5σ RMS threshold based on a continuous 1 second data block to estimate σ RMS on a per channel basis.
Recordings from the MEA plate were performed for 3 consecutive days each week for 4 weeks after plating.During each session, baseline recordings (at 37 °C) were acquired for a duration of 30 minutes followed by a temperature ramp up to 42 °C with the embedded heating plate.The time to 42 °C was 2.5 min and typical decay back to 37 °C was 3.5 min.For the sake of classifying cells over time in response to temperature ramps, we grouped recordings into different classes: "Consistent responders", which includes recordings where the mean ring rate (MFR in Hz) showed elevations from baseline to all 3 temperature ramps for a given week; "responders" were those recordings demonstrating an elevation in at least 1 or 2 of the 3 sessions; "negative responders" were recordings demonstrated spontaneous activity, however there was a decrease in the MFR during temperature ramps; and "inactive electrodes" where the recordings failed to demonstrate spontaneous or temperature ramp-evoked spikes.

Statistical analysis
Statistical analysis was performed using GraphPad Prism v9.3.1 (GraphPad Software, Inc.).Two groups were compared by a Mann-Whitney or Multiple t tests.Comparisons between three or more groups were performed using a one-Way or two-Way ANOVA followed by Bonferroni's, Sidak's or Tukey's multiple comparisons test.Data are presented as mean ± standard error of the mean (SEM) where P values < 0.05 were considered signi cant.

Differentiation of iPSCs into sensory neurons
Multiple iPSC lines were used for functional and molecular characterization of sensory neurons to compare the Anatomic differentiation protocol with the small molecule approach [4].The clinical phenotypes of subjects used in the study are shown in Table 1.iPSCs from all groups were pluripotent by morphology as shown in Fig. S3.These clones were tested for pluripotency markers earlier as described in All iPSC clones were differentiated into peripheral neurons using the Anatomic protocol for 7 days followed by feeding the culture with Anatomic maturation medium until 4-5 weeks.Ctrl1 iPSCs were additionally differentiated with the Chambers protocol for 10 days and MACS sorted for p75 NTR marker followed by maturation for 4 weeks.The schematic outlining of both the protocols is shown in Fig. 1A.
Sensory neurons derived from single cell or clump seeded iPSCs show similar functional properties Human embryonic stem cells (ESCs) and iPSCs survive poorly after individualization (i.e., dissociation and reseeding as single cells) because these cells are more sensitive to treatments and are prone to cell death [31].Stem cells are usually passaged as aggregates during enzymatic dissociation which allows for a long-term expansion without affecting the cell survival and karyotype [32].As iPSC lines are generally maintained as colonies, it is desirable to differentiate them as colonies for greater e ciency and yield to speci c cell types.Most of the differentiation protocols reported in the literature involve single-cell seeding of iPSCs for generation of sensory neurons.Single cell maintenance requires using a ROCK inhibitor to enhance cell survival as longer exposure has shown changes in cellular metabolism [33].We assessed the differentiation potential of the Anatomic protocol with single cell versus clump seeding methods.Seeding at too low density can result in substantial loss of viability.Very large iPSC colonies (200 cells or greater) will affect downstream differentiation processes.We optimized the seeding densities for maximum e ciency.
iPSCs from Ctrl1 were used to investigate if the two seeding methods result in differences in the e ciency to produce neurons, expression for peripheral neuronal marker proteins, and in their resulting AP properties.Phase contrast imaging of the differentiation DIV0 to DIV7 did not suggest any morphological differences between single cell and clump seeding methods (Fig. 1B).Cells were further grown in maturation medium until DIV39.Figure 1C shows phase contrast images of neuronal maturation from DIV8 to DIV34.iPSCs from Ctrl2 were also differentiated with both seeding protocols with comparable morphological appearance to Ctrl1 (Fig. S4).
Immuno uorescence staining across different time points DIV8, 14, 28 and 35 was performed.We checked for Peripherin/Tuj1 marker expression to con rm peripheral lineage of generated neurons.
Immunostaining con rmed expression of peripherin and Tuj1 proteins at DIV8, 14, 28 and 35 of maturation with both the seeding methods (Fig. 1D-E).
Neurons were classi ed as mature or immature based on their ability to generate over-shooting APs [34].
We examined the electrophysiological properties of these neurons at DIV14, 28 and 35 and divided the APs either as mature or immature based on spike overshoot, where mature was de ned as showing an overshoot above 0 mV.We found that immature neurons revealed a wider half-width duration as compared to mature neurons as shown in (Fig. 2A i).We found 100% of neurons patched display mature APs starting at DIV28 of maturation for both clump and single cell seeding methods (Fig. 2A ii).The RMP did not show any signi cant differences over the maturation time (Clump Ctrl1 DIV14 -74.7 ± 4 mV n = 5, DIV28 -74.3 ± 2.8 mV n = 5, DIV35 -72.8 ± 3 mV n = 6, Single cell Ctrl1 DIV14 -68.3 ± 2.3 mV n = 5, DIV28 -72.6 ± 2.5 mV n = 6, DIV35 -71.9 ± 1.7 mV n = 6 p > 0.05) (Fig. 2B and Table 2).Cell capacitance increased with longer maturation time for both single cell and clump seeding protocols, but no signi cant difference was observed between the groups at corresponding days of maturation (Fig. 2C and Table 2).The SCN10A gene encoding the TTXr voltage-gated ion channel Nav1.8 has been functionally characterized in both rodent and human DRG neurons [35].To test for its functional expression, we performed whole-cell voltage-clamp recordings with 500 nM TTX.Voltage protocol and representative TTXr current traces are shown in Fig. 2D i and ii respectively.We found an increase in TTXr current density with maturation time for Ctrl1 (clump seeding) neurons (Fig. 2D iii and Table 3).

Table 3: TTXr current density Ctrl1 and Ctrl2
A-887826 blocked TTXr Na + currents from rat DRG neurons in a state-dependent fashion with an IC 50 of 7.9 ± 0.2 nM for the inactivated state (-40 mV) to an IC 50 of 63.6 ± 0.2 nM when channels were held in a resting state at − 100 mV [29].To identify the functional Nav1.8 ion channel expression in the generated neurons, we perfused 1µM A-887826 in the presence of 500 nM TTX on iPSC-derived sensory neurons at DIV31 of maturation.A single pulse voltage clamp protocol was used from holding potential of -120 mV to -30 mV, applied every 10 s (Fig. S5 i).A-887826 inhibited more than 95% of TTXr currents (n = 2) suggesting the presence of TTXr Nav1.8 currents in the iPSC-derived nociceptors (Fig. S5 ii).
We further evaluated the functional expression of sodium and potassium ion channels using physiological solutions and performed voltage clamp recordings on DIV14 of maturation.When held at -90 mV, both inward Na + and outward K + currents were recorded, indicating functional expression of both Na + and K + ion channels in these sensory neurons (Fig. 2E i and ii).No signi cant difference was observed in peak Na + (Clump Ctrl1 at DIV14: 271.2 ± 19.2, n = 5; Single cell Ctrl1: 288 ± 87.5, n = 3, P ≥ 0.9) or K + (Clump Ctrl1 DIV14 99.2 ± 7.5 n = 5, Single cell Ctrl1 79.5 ± 12.8 n = 3 P ≥ 0.9) current densities (pA/pF) between single cell and clump seeding methods (Fig. 2E iii-iv and Table 4).
Table 4: Sodium and potassium currents Ctrl1 and Ctrl2 We applied depolarizing 1 nA ramp currents over durations ranging from 100-1000 ms to assess ring in response to slower depolarization changes (Fig. 2F i) and classi ed these neurons as tonic or phasic ring.Neurons were considered tonically ring if they generated ≥ 2 APs and phasic for single AP only.
These data suggest that both single cell and clump seeding protocols result in generation of peripheral sensory neurons with almost no difference in the morphological, electrophysiological and marker expression properties of generated neurons.We thus performed further differentiations of all clones used in the study with the single cell seeding protocol.
Anatomic protocol allows the differentiation of a "di cult" iPSC clone derived from a healthy subject Differentiation of iPSCs into sensory neurons, especially when using the Chambers differentiation protocol, suffers from high clone-to-clone variability [10].We have identi ed a speci c iPSC clone derived from a healthy subject (Ctrl2 termed as "di cult clone"), which does not differentiate well with the Chambers protocol.With the Chambers protocol, differentiation results in neuronal cultures with a very low percentage of peripherin-positive neurons (Fig. S1).To understand if the Anatomic protocol results in improved differentiation and more effective generation of sensory neurons with a di cult clone like Ctrl2, we performed a detailed functional and immunocytochemistry analysis on Ctrl2 iPSC-derived neurons.
Phase contrast imaging of the differentiation DIV0 to DIV7 shows generation of immature sensory neurons by DIV7 (Fig. 3A).Cells were further cultured in maturation medium until DIV35.Figure 3B shows phase contrast images of neuronal maturation from DIV8 to DIV34.Immunostaining con rmed the marker expression of peripherin and Tuj1 proteins from DIV8 onwards in these cells (Fig. 3C).
To further con rm the functional properties of these neurons, we performed patch clamp recordings on different days of maturation.Mature APs were detected starting at DIV14, and their percentage increased with maturation time (Fig. 2A ii).RMP was not signi cantly different from DIV14 to 35 (Fig. 2B and Table 2).A signi cant increase in cell size was observed with maturation similar to Ctrl1 iPSC-derived neurons (Fig. 2C and Table 2).Voltage clamp recordings suggest the functional expression of TTXr currents, with an increase in current density recorded from DIV14 to DIV35 (Fig. 2D iii and Table 3).We found a signi cant increase in the Na + and K + current density from DIV14 to DIV28 (Fig. 2F iii-iv and Table 4).
Using ramp stimulation to identify tonic and phasic ring neurons, we observed tonic ring neurons starting DIV28 for Ctrl2 neurons (Fig. 2G iii).
These data suggest that the Ctrl2 iPS cell line, which was di cult to differentiate with the Chambers protocol, displayed robust differentiation towards a peripheral neuronal lineage and generates a dense neuronal network of sensory neurons with the Anatomic protocol.

Chambers protocol results in predominantly tonic ring neurons compared to Anatomic protocol
To understand the functional and immunocytochemical differences between the Anatomic and Chambers protocols, we differentiated blood-derived iPSCs from a healthy subject (Ctrl1) into sensory neurons using both protocols.
Phase contrast imaging shows seeding of Ctrl1 single cells on DIV0 and formation of immature neurons by DIV10 of differentiation with Chambers protocol (Fig. 4A).To enrich the neuronal population towards homogeneous peripheral neuronal cultures, we performed MACS sorting for CD271-expressing cells on DIV10 of differentiation.Neural progenitors obtained were then matured until DIV35.We found signi cant growth of non-neuronal cells as shown in Fig. 4A at DIV18 and DIV35 marked with red arrows, respectively.Young et al., 2014 6 reported the use of antimitotic agents to reduce the number of proliferating non-neuronal cells at the beginning of maturation while using the Chambers protocol.The Anatomic protocol did not show the presence of non-neuronal cells as shown in Fig. 4B at DIV35.
The neurons generated by the Chambers protocol expressed peripherin and Tuj1 from DIV11 onwards (Fig. 4C).Current and voltage clamp recordings showed mature APs starting from DIV14.At DIV28, all recorded neurons showed APs with an overshoot above 0 mV, similar to neurons generated with the Anatomic protocol (Fig. 5A).The RMP was found to be more depolarized at DIV14 as compared to DIV28 and DIV35 for the Chambers protocol.We did not detect a signi cant difference over the same time span with the Anatomic protocol (Fig. 5B and Table 5).We observed an increase in cell size with maturation from both protocols and neurons with the Chambers protocol displaying signi cantly larger cell capacitance (Fig. 5C and Table 5).TTXr current density measured in the presence of TTX (500 nM) suggest the presence of Nav1.8 ion channels in these neurons on DIV28 with no signi cant difference in current density (pA/pF) compared to those neurons generated by the Anatomic protocol (Chambers DIV28: 72.2 ± 12.7, n = 3 vs Anatomic DIV28: 111.9 ± 25.4, n = 6; P = 0.38) (Fig. 5D).With physiological solutions, voltage clamp recordings revealed no signi cant difference in Na + current density (Fig. 5E and Table 6), but we found signi cantly decreased K + current density for neurons differentiated with the Anatomic protocol (Fig. 5F and Table 6).Tonic ring was predominant in the Chambers protocol (Fig. 5G).Ramp stimuli also resulted in increased neuronal ring with the Chambers protocol at DIV35 (Fig. 5H), possibly due to robust K + channel expressions: we have observed that neurons on DIV14 from Chambers protocol show signi cantly higher K + current density as compared to the Anatomic protocol.Both methods resulted in the differentiation and production of mature electrically-active sensory neurons from DIV28.

Nociceptor marker expression and functional characterization of Anatomic Protocol with RealDRG™
Next, we sought to further evaluate the Anatomic protocol by characterizing Anatomic's commercially available iPSC-derived sensory neurons (RealDRG™).We assessed RealDRG™ for nociceptor marker expression using in situ hybridization and their functional pro ling with membrane potential assays, automated patch clamp and multi-well microelectrode arrays.

In situ hybridization of RealDRG™ neurons demonstrates correlation to human DRG gene expression
To evaluate the expression of common human DRG nociceptor markers, multi-plex in situ hybridization was completed using the RNAscope protocol [30].Two separate experiments were completed on RealDRG™ Anatomic Protocol neurons ~ 1 week after plating (DIV14 and 16).In the rst experiment, mRNA for NTRK1 (tropomyosin kinase receptor A, TRKA), TAC1 (preprotachykinin-1), and HCN2 (Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2; HCN2) were probed at DIV14 (Fig. 6A-B).In the second experiment, mRNA for SCN10A (voltage gated sodium channel 1.8; Nav1.8),TAC1, and TRPV1 (transient receptor potential cation channel subfamily V member 1) were probed at DIV16 (Fig. 6C-D).All of these genes are highly expressed in human DRG neurons recovered from organ donors [36].Over 80% of the RealDRG™ expressed NTRK1 and HCN2.Importantly, almost 60% of the Anatomic cells expressed SCN10, a marker of nociceptors.Many of the RealDRG™ (42.8%) also expressed TRPV1 consistent with capsaicin responses seen in Fig. 7F-G and heat sensitivity seen in Fig. 9. TAC1, which is a pre-proprotein that codes for neurokinin A and substance P, was expressed in 7.7% and 11.7% of these cells on DIV14 and DIV16, respectively, demonstrating consistent expression between these two time points, but far lower expression than seen in human DRG neurons [37].
FLIPR Ca 2+ assays show that RealDRG™ respond to a variety of nociceptor stimuli in a 384-well format To evaluate if RealDRG™ might also be amenable to high-throughput uorescence assays, we rst optimised seeding density in a 384-well format and assessed KCl depolarization-induced Ca 2+ responses.
As commonly observed for uorescence assays, higher seeding densities resulted in enhanced signal-tonoise ratios, with 10,000 cells/well performing best (Fig. 7A).We next compared responses to the voltage activated Na + channel activator veratridine (50 µM), the TRPV1 agonist capsaicin (1 µM) and KCl-induced depolarisation in iPSC-derived sensory neurons differentiated using the Anatomic protocol for DIV14, DIV21, and DIV35.Interestingly, KCl-and veratridine-induced responses were similar at DIV15 and DIV21 but decreased in magnitude at DIV35 (Fig. 7B-E), possibly due to the emergence of larger cell clusters following prolonged in vitro differentiation that may interfere with dye loading, while capsaicin-induced responses could only be observed at DIV35 but not at earlier timepoints (Fig. 7F-G).Veratridine-induced responses were inhibited completely by TTX (1 µM; Fig. 7E), consistent with the relatively larger TTX-s component observed in patch-clamp studies, as well as the resistance of the TTX-r Na V 1.8 to modulation by veratridine [38].

RealDRG™ activity can be assessed by automated patch clamp recordings
RealDRG™ were characterized for functional expression of Na + and K + ion channels using APC.
For the cells passing the membrane resistance and cell size lter criteria, average expression levels of voltage gated K + and Na + channels, and AP rings were 90%-97%, 60%-77%, and 60%-67%, respectively (Fig. 8C).We examined the Na + and K + current densities measured at depolarization potential to − 10 mV, Multi-well microelectrode arrays demonstrate the spontaneous activity and temperature sensitivity of RealDRG™ Anatomic protocol RealDRG™ were grown on Axion 48-well (16 electrodes/well) MEAs (Fig. 9A-B).We documented the mean ring rate (MFR) per electrode during each data acquisition session for three days each week for four weeks total starting on DIV14.The active electrode yield (AEY) was calculated based on electrodes whose MFR was 1 spike/min or higher per session.RealDRG™ cells respond robustly to 37to-42 o C temperature changes during all recording sessions (DIV36) (Fig. 9C) consistent with functional TRPV1 receptors which respond to this temperature range.Within a single well (16 total microelectrodes), we observed a variety of ring patterns that change across the 4 weeks in vitro (Fig. 9D).AEY ranged from 77.6 to 22.4% with a mean ± SEM of 55.2 ± 3.7% (n = 384 microelectrodes) with distinct patterns (Fig. 9E).AEY tended to decrease over time but was stable during the three recording sessions each week.We found that the average MFR from all microelectrodes ranged from 0.08 to 0.78 Hz with a mean ± SEM of 0.43 ± 0.04 Hz with the highest and most stable patterns of activity seen during the 4th and 5th week recording sessions (Fig. 9F).Looking at the impact of the 42 o C temperature ramp on modulating the MFR at each DIV, there was a signi cant main effect of temperature (2-WAY ANOVA, p < 0.0001; main effect of temperature x DIV P < 0.0001) with Sidak's multiple comparisons tests showing signi cant differences between baseline and heated at most of the time points (Fig. 9F).Additionally, there was a signi cant effect of DIV time (2-WAY ANOVA, main effect of DIV P < 0.0001).A closer look suggested changes in baseline MFR ("pre-heat") both between weeks and during each week's three consecutive recordings.The difference within weeks was most apparent for weeks 2-4 but stabilized within the week 5 recording sessions.When classifying the cells by week, we found some notable patterns (Fig. 9G).First, most cells responded to the temperature ramp early after plating (week 2).Furthermore, the proportion of cells that responded to the temperature ramp was fairly consistent across weeks and appeared to increase by week 5.For example, during week 4 the combination of "consistent responders" and "responders" (see Methods) reached 85.6%.By week 5, this sum of "responders" and "consistent responders" signi cantly increased to 97.7% (p < 0.0001, t-test).

Differentiation of iPSCs from pain patients
One of the signi cant advantages of iPSC-derived sensory neurons is to understand physiological changes that occur in the disease.We utilized the Anatomic protocol to differentiate iPSCs from two different pain syndromes: SFN and IEM.

Electrophysiological phenotyping of SFN and IEM nociceptors displayed reduced AP rheobase and hyperexcitability
With an e cient generation of sensory neurons from Ctrl1 and Ctrl2 subjects using Anatomic protocol, we next examined the phenotype of iPSC-derived neurons from pain patients for disease modelling.We pooled the data from single cell and clump seeding for Ctrl1 iPSC-derived sensory neurons as we did not nd any differences in their functional properties (Fig. 2).All the patch clamp data for disease modelling for Ctrl1, SFN, and IEM subjects were collected from DIV35-40.All three groups were differentiated once to generate sensory neurons.
Immuno uorescence staining con rmed the expression of peripherin and the pan-neuronal marker Tuj1 at DIV8, 14, and 28 for SFN-derived neurons and at DIV39 for IEM-derived neurons (Fig. S6A-B).Voltage clamp recordings indicate the presence of TTXr currents in IEM and SFN patient-derived neurons (Fig. S7).
To assess the neuronal excitability and AP parameters from iPSC-derived sensory neurons, we performed current-clamp recordings.Cell capacitance and RMP did not show signi cant difference in both patientderived neurons as compared to control group (Fig. 10A-B and Table 7).We found that the patient sensory neurons require signi cantly less current injection to generate APs compared to the control group (Fig. 10C and Table 7).There was a signi cant difference between AP threshold of the patient-derived neurons as compared to the control group (Fig. 10D and Table 6).AHP was much more depolarized for the SFN patient-derived neurons compared to the control group (Fig. 10E and Table 7).
We found a signi cant increase in the number of APs generated in response to ramp current stimuli (500 pA/500 ms) compared to control group (Fig. 10F and Table 7).Patient-derived neurons require signi cantly less time to generate the rst AP in response to slow ramp depolarizations compared to control group indicating increased excitability for both IEM and SFN patient-derived neurons (Fig. 10G and Table 7).Diseased sensory neurons showed a signi cant increase in the number of APs red, indicative of hyperexcitability in response to slower ramp depolarizations (Fig. 10H).We did not observe spontaneously ring neurons with SFN-derived neurons although our recording protocol limited the observation time to only 4 sec.One of 16 IEM-derived neurons showed spontaneous ring.Altogether, electrophysiological phenotyping showed characteristics of elevated excitability in IEM and SFN-derived nociceptors.
Table 7. AP characteristics of Ctrl1, IEM and SFN neurons showed a robust expression of p75 and human natural killer 1 (HNK1) double positive cells indicating a high e ciency of neural crest induction for generation of peripheral sensory neurons.We implemented MACS sorting for p75 marker at DIV10 of differentiation to obtain a more pure neuronal culture.However, one of the control clones still showed non-neuronal cell growth in the culture after MACS sorting (Fig. 4A).Morphologically, the Anatomic protocol does not yield detectable non-neuronal cells with four different cell lines tested for generation of sensory neurons (Fig. 1B-C, Fig. 3A-B).The Anatomic protocol was suitable for differentiating Ctrl2, the "di cult clone."This clone has a signi cantly lower differentiation potential with Chambers protocol in generating peripheral neuronal lineage; we successfully differentiated Ctrl2 with the Anatomic protocol and achieved a dense neuronal network with peripheral neuronal lineage (Fig. 3).Taken together these results indicate that Anatomic protocol shows the potential to e ciently differentiate iPSCs into sensory neurons from multiple cell lines.

Functional characterization: Anatomic Chambers protocol display tonic ring
Conventional differentiation protocols require a long maturation period of 8-10 weeks to yield mature sensory neurons for disease modelling [41,42,20].While the Anatomic protocol produced a purer cell culture, the duration required for maturation was similar to that needed for the Chambers protocol.We performed a detailed functional characterization of two control and two cell lines from pain patients with the Anatomic protocol and compared them with the Chambers protocol for one of the control cell lines.We found that both Anatomic and Chambers protocols resulted in neurons with mature AP characteristics from DIV28 of maturation and expression of TTXr currents from DIV14 onwards indicating presence of nociceptive-like neurons (Fig. 2A and 2D).Pharmacological evaluation indicated the presence of TTXr currents by a selective Nav1.8 blocker (Fig. 2E).We also found that K + current density was signi cantly lower in Anatomic protocol on DIV14 as compared to Chambers protocol, which may have contributed to the higher tonic ring observed with Chambers protocol (Fig. 5H).We also observed increased K + current density with maturation using automated patch clamp recordings (Fig. 8D).We found that both protocols generate mature sensory neurons with peripheral neuronal identity at about the identical time point.We nd a striking difference in the expression of voltage activated K + channel function indicating a higher tonic ring observed with neurons from the Chambers protocol.Future work will assess the subtypes of sensory neurons produced by both differentiation protocols.

In situ hybridization reveals expression of nociceptor markers in RealDRG™
In situ hybridization of RealDRG™ demonstrated both evidence of heterogeneity in the Anatomic cells and similarity between Anatomic protocol cells and human primary nociceptors.First, heterogeneity of nociceptive markers in Anatomic cells indicates that the cells may be useful as a model to explore human DRG physiology as a whole.Second, we nd evidence for good, although not perfect, expression patterns in the Anatomic protocol RealDRG™ cells compared to human DRG.The majority of neurons express SCN10A and many of those also express TRPV1 (~ 50%), similar to human neurons, although with a smaller population that is TRPV1-positive (human DRG ~ 75% TRPV1) [36].Only 15-20% of neurons generated with Chambers protocol were responsive to capsaicin [20].Also consistent with human DRG neurons [36], most RealDRGs express NTRK1, which is a major difference that distinguishes human nociceptors from mouse nociceptors where only half of the cells express NTRK1 [37].HCN2 expression was similar to what is observed in the native human DRG [43].While these preliminary ndings require further investigation to characterize these populations more thoroughly, the existing data suggest that the neurons derived from the Anatomic protocol can be binned into actions of particular subsets of neurons that may be informative in making decisions about human nociceptors.
FLIPR, MEA, automated patch clamp recordings for high-tomedium throughput pharmacological screening A major advantage of human iPSC-derived sensory neurons is the ability to generate, at least in principle, unlimited numbers of cells, which opens the possibility of adapting these cellular model systems to highthroughput drug discovery efforts.In FLIPR Penta assays adapted to 384-well format, we observed particularly robust depolarisation-induced responses that are likely mediated by voltage-gated Ca 2+ channels.In contrast, responses to capsaicin were relatively small and would likely require further optimisation of assay conditions.Given the tendency of cells differentiated for more than 4-5 weeks to form cell clusters, this could include bulk differentiation and subsequent dissociation/replating for highthroughput assays, similar to our approach for automated patch-clamp electrophysiology.While we did not observe functional responses to menthol or AITC (Fig. 7H), suggesting that Anatomic iPSC-derived sensory neurons represent a subset of the functionally diverse types of DRG neurons found in primary culture, our transcriptomic analysis suggests that these cells can be adapted for functional assays assessing responses of a range of therapeutically relevant targets.In addition to FLIPR assay for high throughput recordings, we have shown functional recordings of Na + and K + channels in RealDRG™ with automated patch clamp recordings (Fig. 8).We observed success rates of > 40% over the maturation period starting DIV16, 21, 28, and 35 days (Fig. 8C).The success rate decreased with increasing maturation indicating dense neuronal network formation of iPSC-derived neurons making it di cult to obtain single cells after dissociation.We also observed TTXr currents in these neurons at DIV21, 28 and 35 of maturation (Fig. 8J).
Finally, we utilized MEAs to evaluate ring of Anatomic protocol RealDRG™ across time and responses to heat ramp stimuli.MEAs in plate form provide a valuable read-out (i.e., ring) for drug screening efforts.
We nd that RealDRG™ are spontaneously active with good stability during a single week and variability in active yield between weeks.For drug screening this would indicate that testing could occur within a week in a single plate but that week-to-week comparisons might be more di cult to interpret.The RealDRG™ respond to temperature ramps, again indicating the functional presence of heat activated channels such as TRPV1.From a drug screening perspective, such phenotypic responses of RealDRG™ would be useful for distinguishing between drugs that inhibit all sensory neurons versus only those cells with heat sensitivity.
Taken together all three automated/semi-automated platforms provide a valuable approach in characterizing various ion channels from iPSC-derived sensory neurons to facilitate drug discovery and development of novel targets.

Electrophysiological phenotype indicates hyperexcitability with sensory neurons from pain patients
We established the generation of functional sensory neurons with Anatomic protocol having mature AP properties from DIV28 of maturation.With an e cient generation of sensory neurons from Ctrl1 and Ctrl2 (di cult clone) subjects using Anatomic protocol, we next examined the phenotype of iPSC-derived sensory neurons from pain patients for disease modelling.We investigated sensory neurons derived from two patients suffering from IEM and SFN disorders.Cellular excitability of SFN patient-derived nociceptors displayed a signi cantly increased excitability: more spikes and more active electrodes when recorded with MEAs [23].We found a signi cant reduction in the current threshold for the patient-derived neurons compared to control group (Ctrl:121 ± 16.3 pA, IEM: 31.1 ± 3.9, SFN: 47.4 ± 5.3 p < 0.05) (Fig. 10C).In addition, we found a similar hyperexcitability phenomenon to evoked stimuli with manual patch clamp recordings in response to ramp stimuli (Fig. 10H).IEM neurons derived by the Anatomic protocol also displayed higher excitability to electrical stimuli as reported with Chambers protocol (Fig. 10H).The AP threshold remained unaltered in SFN-derived nociceptors with Chambers protocol [23].
It has been reported that L554P/Nav1.8 mutation causing SFN disorder results to signi cant reduction in current threshold after expression of L554P mutation in DRG neurons and no change in RMP was observed [44].Similarly, the pathogenic variant found in SCN10A (c.5116A > G) identi ed in a pain patient revealed that mutant channels decrease current threshold and increase the ring frequency of evoked APs when transfected into small rodent DRG neurons [45].We found similar results with SFN-derived neurons using Anatomic protocol.

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Figure 5 Comparison
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Table 6 :
Sodium and potassium currents Chambers vs Anatomic Control ΔF/F 0 2.97 ± 0.57, IL6 ΔF/F 0 3.03 ± 0.49; p > 0.05), capsaicin (1 µM; Control ΔF/F 0 0.70 ± 0.09, IL6 ΔF/F 0 0.62 ± 0.12; p > 0.05), or the Piezo1 agonist Yoda1 (100 µM; Control ΔF/F 0 0.36 ± 0.08, IL6 ), but observed no difference in response to either KCl (Control ΔF/F 0 4.28 ± 0.77, IL6 ΔF/F 0 4.35 ± 0.30; p > 0.05), veratridine (50 µM; [37,49] molecular characterization of iPSC nociceptors with single cell sequencing should be considered for identi cation of speci c neuronal subtypes and comparison to existing human nociceptor datasets[37,49].for publication manuscript.B.J.K., J.P., T.J.P., G.D. Conceived the study, planned and designed experiments, interpreted the data, wrote and critically revised the manuscript.A.L. Conceived the study, planned and designed the experiments, interpreted the data, and wrote and critically revised the manuscript.All authors read and approved the nal manuscript. Recently Saito-Diaz et al., 202147reported generation of three sensory neuronal subtypes-nociceptors, mechanoreceptors, and proprioceptors by day 20 of maturation at a ratio of approximately 2:1:1, correlating with the relative sensory neuronal distribution in the human DRG in vivo[48].It is possible that subtype speci c neurons generated with Anatomic protocol might not represent the molecular diversity of DRG sensory neurons found in vivo although many of our results suggest that some important nociceptor subtypes are represented.Ethics approval was obtained from the ethics committee of the Medical faculty of the RWTH reference number EK243/18 (2019 and amendment 2022), title "Generierung von humanen Zellmodellen sensorischer Neuropathien" ("generation of human cellular models for sensory neuropathies").